Project Summary Chimeric RNAs are transcripts in which the nucleotide sequence reflects the sequence of two different parental genes. They are typically thought to arise from gene fusions which form via chromosomal rearrangement events. However, we propose that the inverse may be true: that chimeric RNAs may preempt and contribute to the formation of gene fusion. Chromosomal rearrangements are dependent upon the pre- existence of DNA double-stranded breaks (DSBs) and result from the erroneous repair of free DNA ends. Rearrangements occur more frequently between breaks in close interphase genomic proximity and breaks with homology at free DNA ends. DNA breaks occur non-randomly and tend to cluster in loci termed breakpoint cluster regions (BCRs). While some BCRs are consistent between cell types, chromosomal rearrangements tend to be more cell/tissue-type specific. This contrast indicates that translocation induction from existing DSBs is influenced by cell type specific factors. Chimeric RNAs exhibit many of these qualities, which increase the likelihood for gene fusions. They possess homology spanning the translocation junction, have significant tissue-specificity, and are proposed to exist in close proximity to its parental gene loci. Most importantly, we have discovered chimeric RNAs which exactly match transcripts from neoplastic gene fusion events in normal cells without corresponding changes to the genome. Thus, these chimeric RNAs precede the translocation, and may play a role in their induction. We have two established models in which to test this concept. First, we have a non-cancer thyroid cell line in which we have previously characterized DSB and RET/CCDC6 translocation generation in response to chemical treatment. No RET/CCDC6 chimeric transcript has been detected in untreated cells. Thus, ectopic addition of exogenous chimeric RET/CCDC6 provides a robust model to assess the effect of the chimeric transcript on translocation frequency. Second, we have characterized PAX3-FOXO1 chimeric RNA expression throughout mesenchymal stem cell myogenesis. PAX3- FOXO1 chimeric RNA is exclusively expressed or not expressed at discrete time points through MSC differentiation. Therefore, DSB generation at these time points allows for assessment endogenous chimeric RNA's effect on translocation frequency. This proposal would be the first to exhibit a role of chimeric RNA templates in gene fusions and would offer novel insight into the risk factors and specificity of chromosomal rearrangement. Implication of chimeric transcripts in gene fusions would have significant impacts on basic research into mechanisms of chromosomal rearrangement and DNA repair, and could translate to rapid development of preventative screening and detection measures for individuals at risk for these translocations.